One of the major challenges of HlV-1 vaccine development is the elicitation of broadly neutralizing antibodies (bNAbs) against HIV Env. The recent isolation of several bNAbs from HIV-infected individuals demonstrates that the human B cell repertoire can generate bNAbs targeting the conserved Env region. However, there is still a tremendous knowledge gap regarding how the broad responses are elicited during chronic HIV-1 infection and, additionally, how these compare to the much more limited responses elicited by Env vaccination. To fill this information gap, we propose to define and improve the elicitation of neutralizing antibody responses toward the most functionally conserved and accessible element ofthe HIV-1 Env complex, including the receptor CD4 binding site (CD4bs). Previously, we developed a multicolor Env epitope-specific B cell sorting and RT-PCR strategy to analyze the memory B cell compartment of HIVinfected individuals, which led to the isolation of CD4bs potent and bNAb, VRCOl, which neutralizes >90% of circulating primary viruses. We adapted this epitope-specific memory B cell sorting strategy to extend our analyses to gain insight of Env B cell response during and following Env immunogen vaccination into nonhuman primates (NHPs). Accordingly, the aims of this Project 3 are summarized as follows. In Aim 1 we will develop envelope-specific, including CD4bs-specific memory B cell isolation and monoclonal antibody cloning from Env immunogen vaccinated NHP animals and design new and novel probes specific for bNAb isolation. In Aim 2, we will characterize the Env-specific monoclonal antibody specificities with state ofthe art binding and characterization assays. In Aim 3, we will investigate the role, composition, and evolution pathway ofthe Env-specific IgM memory B cell compartment, a subset of memory B cells, which recently has been shown to play an important role in the long term B cell response.